Shift in Conformational Equilibrium Induces Constitutive Activity of G

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B: Biophysical Chemistry and Biomolecules

Shift in Conformational Equilibrium Induces Constitutive Activity of G-Protein Coupled Receptor, Rhodopsin Ryo Maeda, Michio Hiroshima, Takahiro Yamashita, Akimori Wada, Yasushi Sako, Yoshinori Shichida, and Yasushi Imamoto J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02819 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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The Journal of Physical Chemistry

Shift in Conformational Equilibrium Induces Constitutive Activity of G-Protein Coupled Receptor, Rhodopsin Ryo Maeda,†,‡ Michio Hiroshima,‡,|| Takahiro Yamashita,† Akimori Wada,§ Yasushi Sako,‡ Yoshinori Shichida,†,⊥ and Yasushi Imamoto*,† †

‡

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan,

Cellular Informatics Laboratory, RIKEN, Wako, Saitama, Japan, ||Laboratory for Cell Signaling Dynamics, RIKEN Quantitative Biology Center, Suita, Osaka, Japan, §Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe, Hyogo, Japan, ⊥Research Organization for Science and Technology, Ritsumeikan University, Kusatsu, Shiga, Japan.

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ABSTRACT:

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Constitutively active mutants (CAMs) of G-protein coupled receptors (GPCRs),

cause various kinds of diseases. Rhodopsin, a light-absorbing GPCR in animal retina, has retinal as an endogenous ligand; only very low levels of activation of G-protein can be obtained with the ligand-free opsin. However, the CAM of opsin activates G-protein much more efficiently than the wild type but the mechanism underlying this remains unclear. The present work revisits the constitutive activity of rhodopsin from the standpoint of conformational dynamics. Singlemolecule observation of the M257Y mutant of bovine rhodopsin demonstrated that the switch between active and inactive conformations frequently occurred in M257Y opsin, and frequent generation of the active state results in the population shift towards the active state, which accounts for the constitutive activity of M257Y opsin. Our findings demonstrate that the protein function has a direct connection with the structural dynamics.


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INTRODUCTION G protein coupled receptors (GPCRs) are ubiquitous cell surface receptor proteins with a common seven-transmembrane domain structural motif. They respond to specific ligands as stimuli and activate their cognate G proteins, which initiate the signal transduction cascade in the cell. GPCRs act as molecular switches and should not exhibit basal activity in the absence of stimuli to improve the sensitivity by eliminating the noise. It is especially essential for a lightabsorbing GPCR in retinal rod cells, rhodopsin, responsible for the single photon response of the cell. However, it is well known that some mutations of GPCRs result in inherent basal activity16

. These mutants are called constitutively active mutants (CAMs), which exhibit significant G

protein activation effect in the absence of ligand. So far, several CAMs have been identified through pathological analysis as well as mutational characterization of GPCRs. Characterization of CAMs is important because CAMs often cause diseases, e.g. CAM of rhodopsin causes autosomal dominant retinitis pigmentosa or night blindness7. One of well-known CAMs of rhodopsin, M257Y, shows high constitutive activity and is characterized as an experimental model of CAM of GPCRs3, 8-10. While most GPCRs are activated by diffusible ligands, the activity of rhodopsin is regulated by the chromophore retinal, which acts as an endogenous ligand. In the dark state, retinal is in 11cis configuration and acts as an inverse agonist. On photon absorption, the chromophore is isomerized to the all-trans form, resulting in the formation of a G-protein activating intermediate called metarhodopsin II (Meta-II), where retinal acts as an agonist.

G-protein activation

efficiency of apoprotein opsin (ligand-free form) is ~100 times lower than that of agonist-bound form (Meta-II) in wild type rhodopsin, but that of M257Y opsin is ~30 times greater than that of wild type opsin. The high G-protein activation efficiency of CAM opsin can be explained by the


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altered opsin conformation having relatively high Gt activation efficiency, or the generation of small amount of opsin having Meta-II-like conformation in the ensemble11-14. While crystal structures of CAMs of rhodopsin in ligand-free form have not been available, previous study on the structural dynamics of CAM of rhodopsin using fluorescence quenching supports the latter10. However, it is possible that these two factors synergistically contribute to the constitutive activity. Therefore, direct and quantitative observation of interconversion between active and inactive conformations is essential to address this controversy. Recently, we have demonstrated that the single-molecule analysis using total internal reflection fluorescence microscopy (TIRFM) is a powerful tool to directly monitor the physiologically relevant conformational changes of rhodopsin15. Unlike bulk measurements, single-molecule analysis of individual molecules provides direct information about the duration time in each conformation, and statistical analysis provides information about the kinetics of conformational changes. Here, we labeled M257Y with the Alexa594 fluorophore at Cys316 in the cytoplasmic Helix VIII to probe the conformational change at the single-molecule level. We conducted single-molecule fluorescence microscopy of Alexa594 in the dark states, photoactivated states, and opsin states (without retinal) of wild type and M257Y to address the mechanism of constitutive activity of opsins and GPCRs.


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MATERIALS AND METHODS

Sample Preparation.

Genes of wild type, C140S (WTC140S) and C140S/M257Y

(M257YC140S) of bovine rhodopsin were transiently transfected into HEK293T cell lines16-17. After collection of cells, pigments were regenerated by adding 11-cis-retinal to the cell membranes. For the single-molecule fluorescence measurements in the dark state, the pigments were regenerated with non-isomerizable 11-cis-retinal in which C11=C12 bond is locked in the cis configuration by a 7 membered ring (7m-Ret) to generate fluorescence of Alexa594 without photoreaction of rhodopsin chromophore18-19. Pigments were solubilized by OG/asolectin buffer (60 mM n-octyl-β-D-glucoside, 1 mg/mL asolectin, 50 mM HEPES, 140 mM NaCl, pH 7.5), and adsorbed to rho1D4 (anti-bovine rhodopsin monoclonal antibody) immunoaffinity beads. After washing with OG/asolectin buffer and labeling buffer (60 mM n-octyl-β-D-glucoside, 1 mg/mL asolectin, 5 mM MES, 50 mM HEPES, pH 7.5), 5 times molar excess, over rhodopsin, of Alexa594 maleimide (Molecular Probes) dissolved in DMSO was added to the bead suspension and incubated at 4°C overnight. Then the beads were extensively washed by labeling buffer and OG/asolectin buffer, and the labeled pigments were eluted with 8-meric peptide derived from the C-terminal amino acid sequence of bovine rhodopsin. Typically, the molar ratios of pigment and Alexa594 were estimated to be 1:0.9 for WTC140S and 1:1.2 for M257YC140S. To reconstitute the pigments into the liposome, purified pigments in OG/asolectin buffer (molar ratio of pigment and asolectin was 1:1300) were dialyzed against 500 volumes of dialysis buffer (50 mM MES, 140 mM NaCl, pH 6.0) at 4 °C in the dark for 36 h with six buffer exchanges. Opsin samples were prepared by photobleaching of pigments in the presence of 50


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mM hydroxylamine. For control, the WTC140S sample was put onto the boiled water for 5 min to denature rhodopsin. G protein transducin (Gt) was isolated from bovine retinas20-21. Rod outer segment membranes isolated from 200 bovine retinas were suspended in ROS buffer (10 mM MOPS, 30 mM NaCl, 60 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, pH 7.5) and exposed to a fluorescent desk lamp for 30 min so that rhodopsin is converted into Meta-II to form the complex with Gt. After washing the membrane three times with Buffer A (5 mM Tris/HCl, 0.5 mM MgCl2, 1 mM dithiothreitol, pH 7.0) containing 0.2 mM EDTA, Gt was released from the membrane by adding Buffer A containing 0.1 mM GTP and the soluble fraction was collected by centrifugation. Extraction by GTP was repeated three times. Gt was further purified by DEAE Toyopearl 650S chromatography (Toso).

Bulk Spectroscopy. UV-visible absorption spectra were recorded using a Shimadzu UV2450 spectrophotometer. Fluorescence time courses were recorded with a Shimadzu RF-5300PC spectrofluorometer.

The optical cell holder was temperature-controlled.

The sample was

irradiated with light generated by a 1 kW tungsten halogen lamp and passed through a glass cutoff filter (Y52, Toshiba). Fluorescence of Alexa594 was generated by a 590 nm excitation beam attenuated by a glass cut-off filter (R61, Toshiba) and monitored at 630 nm.

G Protein Activation Aassay. The activation of Gt was assessed by GDP/GTPγS exchange using a radionucleotide filter-binding assay21. All procedures were carried out at 0 °C. Reaction mixtures (~125 nM pigment, 600 nM Gt, 50 mM MOPS, 140 mM NaCl, 2.25 mM MgCl2, 1 mM DTT, 2 µM GDP, pH 7.0) were kept in the dark or irradiated with light generated by a 1 kW tungsten halogen lamp and passed through a Y52 filter for 30 s. The GDP/GTPγS exchange


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reaction was then initiated by the addition of [35S]GTPγS (final concentration, 1 µM). After incubation for a given time in the dark, an aliquot (20 µL) of the reaction mixture was mixed with 200 µL of stop solution (20 mM Tris/HCl, 100 mM NaCl, 25 mM MgCl2, 1 µM GTPγS, and 2 µM GDP, pH 7.4), and was immediately filtered through a nitrocellulose membrane to trap [35S]GTPγS bound to Gt. The amount of bound [35S]GTPγS was quantitated by a PerkinElmer Tri-Carb 2910TR liquid scintillation counter.

Single-Molecule Fluorescence Measurements.

Just before single-molecule fluorescence

measurements, the liposome suspensions containing WTC140S or M257YC140S were centrifuged to remove aggregates that resulted in very bright fluorescence spots in the TIRFM images. Supernatants were supplemented with 2-mercaptoethanol and Trolox (6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid) at the final concentrations of 0.5% (w/v) and ~1 mM, respectively, to suppress photobleaching of Alexa594.

The supernatants were put into the

sample dish with cover glass on the bottom. Fluorescence of Alexa594 adsorbed to the cover glass was observed using the TIRFM composed of an inverted microscope (TE 2000, Nikon) with a 60× oil-immersion objective (ApoTIRF 60× 1.49 NA, Nikon)15 and 4.0× relay lens. Fluorescence of Alexa594 was generated by 559-nm laser (WS-0559-050, 50 mW, NTT Electronics). The energy of the laser was reduced by 75% with neutral density filters. The images were acquired using an EMCCD camera (C9100-13, ImagEM, Hamamatsu) with a 345× EM gain. The 512×512 pixel images were recorded with a time resolution of 20 frames/s and a magnification of 67 nm/pixel. The sample dish was maintained at 6°C with an air-cooling device (Peltier-4, Taiei Denki). All sample manipulations were performed under dim red light.


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Data analyses. Time course of fluorescence intensity was obtained from TIRFM images by using ImageJ. Fluorescence intensity steps were detected by a laboratory-written analytical program based on a hidden Markov model22-23, developed using LabVIEW software (National Instruments)15.

All statistical and kinetic analyses were performed with IGOR Pro 6.3

(WaveMetrics).


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RESULTS We first optimized the methods to label rhodopsin expressed in the cultured cells with Alexa594, which is sensitive to the conformational changes.

In order to exclusively label

Cys316 with Alexa594, Cys140 (another chemically modifiable cysteine residue in bovine rhodopsin24) was replaced by Ser in both the wild type (WTC140S) and M257Y mutant (M257YC140S). Cys316 was labeled with Alexa594 maleimide during the purification using immunoaffinity beads, and these pigments were incorporated into asolectin-liposomes by dialysis. To assess the impact of C140S mutation, Gt activation efficiency of WTC140S and M257YC140S was measured by a conventional filter binding assay using radioactive GTPγS (Figure S1). In the photoactivated state (Meta-II), WTC140S and M257YC140S exhibited similar and significant GDP/GTPγS exchange rate. The inverse agonist (11-cis-retinal) suppressed the Gt activation in the dark state.

However, in the ligand-free form (opsin), the Gt activation efficiency of

M257YC140S was 27-fold greater than that of WTC140S. These results were consistent with those of the previous work for wild type and M257Y3. On irradiation of WTC140S, fluorescence of Alexa594 was increased by 30%, which is derived from the formation of Meta-II in the active (open) conformation (Figure 1a, red). In the presence of hydroxylamine, fluorescence increase was immediately followed by the decrease, indicating that WTC140S opsin is mainly in the inactive (closed) conformation (Figure 1a, blue). However, irradiation of M257YC140S increased fluorescence by only 20%. Since G-protein activation efficiencies as well as crystal structures of photoactivated states are similar between wild type and M257Y rhodopsin3, 9, it is likely that fluorescence intensities of Alexa594 bound to the photoactivated states of WTC140S and M257YC140S are comparable. Therefore, it is likely that


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fluorescence intensity of M257YC140S in the dark state is greater than that of WTC140S. This is consistent with the finding that the environment around Cys316 in the dark state M257Y is similar to that of Meta-II shown by the EPR experiment using the nitroxide side chain25. It should be noted that hydroxylamine did not lower the fluorescence intensity of M257YC140S (Figure 1a), indicating that the fluorescence intensity of M257YC140S opsin was greater than that of WTC140S opsin. Next, the TIRFM images of liposomes containing Alexa594-labeled WTC140S or M257YC140S were recorded every 50 ms, which clearly showed relatively uniform fluorescence spots (Figure 1b). The fluorescence intensity of each spot was determined by calculating the average intensity of 12×12 pixel region. The fluorescence time course of each spot is shown by plotting the fluorescence intensity against time (Figure 1c), and the histograms of maximal fluorescence intensity of the spots were constructed (Figure S2). They were fitted with the sum of two Gaussian functions assuming that the fluorescence spots contain one or two Alexa594 molecules15 (Eq. S1), where ~90% of spots were derived from a single Alexa594 molecule. To exclude the fluorescence spots containing two or more Alexa594-labeled rhodopsin molecules, the fluorescence spots whose intensity is within the full width at half maximum of the main distribution (intensity of a single Alexa594 molecule, blue thick line in Figure S2) were subjected to further analysis. In the time course of the fluorescence intensity (Figure 1c), both WTC140S and M257YC140S showed stepwise fluorescence changes between high-fluorescence state (Fhigh) and lowfluorescence state (Flow), and eventual fluorescence decrease to zero derived from bleaching of Alexa594. The fluorescence change points were detected using laboratory-developed program based on the hidden Markov model (Figure 1c, smooth orange/magenta/cyan lines)15, 22-23. We


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first analyzed the frequency and amplitude of conversion between Fhigh and Flow (Figure 1c, orange). The histograms for the change in the fluorescence intensity (∆I), which was normalized relative to the maximal fluorescence intensity of each spot, are constructed for dark states, photoactivated states, and opsins (Figures 2a and 2b). For the measurements in the dark state, the pigments were regenerated with 11-cis-locked retinal15, mostly contained Meta-II at pH 6.0.

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, and the photoactivated state

Opsin samples were prepared by photobleaching of

pigments in the presence of 50 mM hydroxylamine.

As a control experiment, the same

measurements and analyses were carried out using WTC140S samples denatured by boiling (Figure 2c). The histogram of the denatured sample was fitted with a mirror-imaged distribution, whereas that of photoactivated state was broad and fitted with the combination of two sets of mirror-imaged distributions. The mirror-imaged distribution observed in the denatured sample reflects a noise component (NC, blue line) derived from the false-detection of fluorescence change points in our experimental setup and/or analytical algorithm15. Another distribution observed in the photoactivated sample shows the signal component (SC, green line) derived from the conformational switch between Flow and Fhigh. Assuming that NC was included regardless of the state of the sample, all the histograms in Figure 2 were global-fitted with the combination of mirror-imaged normal distribution for SC and lognormal distribution for NC (Eq. S2), where the parameters for the NC were global (Supporting Information). In addition to the photoactivated state, SC was observed in opsin, where SC of M257YC140S was greater than that of WTC140S (Figures 2a and 2b). It is consistent with the previous study that M257Y mutation lowered the energy barrier between inactive and active conformation of opsin10. In Figure 2d, the amplitudes of SC of the dark states, photoactivated states, and opsins of WTC140S and M257YC140S were compared. Fluorescence changes in M257YC140S opsin occurred


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more frequently than in WTC140S opsin, while the difference between WTC140S and M257YC140S was not significant in photoactivated state. In M257YC140S, frequencies of fluorescence changes in photoactivated state and opsin were comparable, which was consistent with the bulk fluorescence measurement (Figure 1a). It should be noted that a small distribution of SC was also observed for the dark state of M257YC140S. Figure 2d suggests that conformational dynamics in opsin state is notably affected by CAM. To assess the kinetics of the conformational change, the histograms of the probability distribution of duration times in Fhigh (Figure 1c, magenta) and Flow (Figure 1c, cyan) were constructed for photoactivated states and opsin states ( f high → low (t ) and respectively). In addition, the histograms ( f high → low (t ) and

f low → high ( t ) )

f low → high ( t )

in Figures 3a-3d,

of the denatured sample was

also constructed to examine the kinetics of fluorescence changes by NC (Figure 3e). While it is essential to compare the kinetic parameters of interconversion between Fhigh and Flow in WTC140S opsin and M257YC140S opsin, it was difficult to estimate the rate constants of SC from the duration time histograms because significant amount of fluorescence changes was derived from NC in opsin states (Figures 2a and 2b). This means that the probability distribution of duration times is the sum of exponential functions expressing the interconversion between Fhigh and Flow (SC) and false detection (NC), where the latter is significantly greater than the former. To analyze the kinetics involving SC and NC, we generated an equilibrium model in which Fhigh and Flow are composed of two substates showing relatively high and low fluorescence intensity (H and H' for Fhigh and L and L' for Flow, respectively) (Figure S3). In the denatured sample, fluorescence changes are derived only from NC between D and D'.

In this scheme, the

fluorescence conversions for H ↔ H’, L ↔ L’, and D ↔ D’ are assumed to be derived from stochastic false detections (NC) (Supporting Information).


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Based on this model, the probability distributions of duration times before fluorescence decrease ( f high → low (t ) ) or increase ( f low → high (t ) ) were expressed using the rate constants for SC and NC (Eqs. S30-S33). Using these equations, the duration time histograms were global-fitted (Figure 3 and Table S1). This analysis was validated by calculating the ratio of SC and NC using these rate constants, which agreed with the ratio of SC and NC in difference intensity histogram (Figure 2) (Figure S4 and Table S2). The rate constants of conformational change from Fhigh to Flow (kS1) and that from Flow to Fhigh (kS2) are shown in Figure 3f. The kS2 of M257YC140S opsin (0.42 s-1) was about seven times greater than that of WTC140S opsin (0.06 s-1), while difference in kS1 of the two was relatively small (0.38 and 0.99 s-1). These results imply that the population of active state of M257YC140S opsin is increased mainly by the frequent formation of active conformation (Fhigh), rather than the stabilization of active conformation. The fraction of Fhigh was calculated to be 0.521 and 0.056 for M257YC140S opsin and WTC140S opsin, respectively, implying that M257Y mutation causes the decrease in ∆G between Flow and Fhigh by 8.2 kJ/M. This population shift (~10 times) is consistent with the difference in Gt activation efficiency between WT opsin and M257Y opsin3, while it is different from the results of previous bulk fluorescence measurement in which M257Y mutation does not significantly alter the stable population of active conformation of opsin in nanodiscs10.


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DISCUSSION We have previously shown that light-induced generation of the active conformation of rhodopsin could be monitored at the single-molecule level by measuring the fluorescence of Alexa594 immobilized at Cys316. The inverse agonist (11-cis-retinal) shifts the equilibrium toward the inactive conformation, whereas the agonist (unprotonated all-trans-retinal) shifts it toward the active conformation15. A similar observation has been reported for β2-adrenergic receptor, a typical GPCR, in which conformational equilibria between active and inactive states are modulated by the species of diffusible ligands26.

Here, this concept was extended to

constitutive activity of ligand-free opsin. While previous studies have shown that wild type opsin forms Meta-II-like conformation at acidic pH27, our single-molecule observation directly detected the formation of Meta-II-like conformation of opsin at physiologic pH15, where Meta-II-like conformation is minor. By using this technique, we successfully showed that conformational changes occurred more frequently in M257YC140S opsin (CAM) than in WTC140S opsin. In addition, our kinetic analysis found that the elevated rate constant of the conversion from inactive to active conformation (Flow to Fhigh) results in the shift of conformational equilibrium toward the active state in CAM. Quantitative agreement between this population shift (~10 times) and the difference in Gt activation efficiency between WT opsin and M257Y opsin (~30 times) implies that the constitutive activity is mainly accounted for by the population shift mechanism, which is essentially similar to the ligand-induced activation of native rhodopsin in which the frequent generation of active conformation in Meta-II state results in the accumulation of active conformation15 (Figure 4). It should be noted that although the G-protein activation in the dark state of M257Y was not detected, infrequent generation of Fhigh was observed in our single-molecule measurements. This


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discrepancy might be derived from the position of fluorescent Alexa594 which reports the conformational changes. Although Alexa594 bound to Cys316 on helix VIII is sensitive to the environmental change caused by rhodopsin activation28, this site is distant from TM610, 29, major site of conformational change. When light is absorbed by the visual pigments in the cells, visual pigments are bleached to form opsins. Constitutive activity of opsin causes the elevation of the background level of Gt activation. This phenomenon is called bleaching adaptation, and results in the desensitization of the cell.

Therefore, constitutive activity of opsin should be lowered to improve the

photosensitivity of the visual cells. In addition, M257Y has the ability to take up all-trans-retinal as an exogenous ligand to activate Gt3, 8. Met257, which is widely conserved in rhodopsins as well as in cone visual pigments, suppresses not only the constitutive activity but also the uptake of the agonist all-trans-retinal by modulating the conformational dynamics, which is essential to lower the dark noise. It is established that vertebrate rhodopsin has two major ionic locks that maintain inactive conformation in the dark state30. One is between the protonated Schiff base of the chromophore and counterion Glu113, and the other is between cytoplasmic Arg135 and Glu247. In opsin, the chromophore ionic lock is replaced by the salt bridge between Glu113 and Lys296. Since perturbation of the cytoplasmic ionic lock by M257Y mutation dramatically alters both the Gprotein activation efficiency and conformational dynamics in ligand-free opsin state, the cytoplasmic ionic lock would be strongly coupled with Glu113-Lys296 ionic lock in opsin. M257Y mutation would disrupt Glu113-Lys296 ionic lock and destabilize the inactive form, resulting in the ready generation of active conformation (Figure 4).


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CONCLUSION The constitutive activity of M257Y opsin is explained by the shift in conformational equilibrium toward the active conformation. It is essentially consistent with the ligand-induced activations of GPCRs15,

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, indicating that both the constitutive activity and ligand-induced

activity of GPCRs are understood in the same framework of population shift (Figure 4). Since CAM of GPCRs often causes diseases, our findings suggested that dysregulation of protein dynamics has a direct connection to diseases development.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Characterization of WTC140S and M257YC140S; Fluorescence intensity of each fluorescent spot in single-molecule images; analysis of the frequency of fluorescence changes; kinetic analysis; validation of the kinetic analysis (PDF).

AUTHOR INFORMATION *Corresponding Author e-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We are grateful to Prof. Robert S. Molday of the University of British Columbia for providing the hybridoma that produces rho1D4 antibody. This work was supported by JSPS KAKENHI Grant Numbers JP16K07319 and JP16H02515.


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(10) Tsukamoto, H.; Farrens, D. L. A constitutively activating mutation alters the dynamics and energetics of a key conformational change in a ligand-free G protein-coupled receptor. J Biol Chem 2013, 288, 28207-28216. (11) Samama, P.; Cotecchia, S.; Costa, T.; Lefkowitz, R. J. A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model. J. Biol. Chem. 1993, 268, 4625-4636. (12) Lefkowitz, R. J.; Cotecchia, S.; Samama, P.; Costa, T. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol. Sci. 1993, 14, 303-307. (13) Robertson, M. J.; Dougall, I. G.; Harper, D.; McKechnie, K. C.; Leff, P. Agonistantagonist interactions at angiotensin receptors: application of a two-state receptor model. Trends Pharmacol. Sci. 1994, 15, 364-369. (14) Leff, P. The two-state model of receptor activation. Trends Pharmacol. Sci. 1995, 16, 8997. (15) Maeda, R.; Hiroshima, M.; Yamashita, T.; Wada, A.; Nishimura, S.; Sako, Y.; Shichida, Y.; Imamoto, Y. Single-molecule observation of the ligand-induced population shift of rhodopsin, a G-protein-coupled receptor. Biophys. J. 2014, 106, 915-924. (16) Nathans, J. Determinants of visual pigment absorbance: role of charged amino acids in the putative transmembrane segments. Biochemistry 1990, 29, 937-942. (17) Imai, H.; Terakita, A.; Shichida, Y. Analysis of amino acid residues in rhodopsin and cone visual pigments that determine their molecular properties. Methods Enzymol. 2000, 315, 293312.


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(18) Akita, H.; Tanis, S. P.; Adams, M.; Baloghnair, V.; Nakanishi, K. Non-bleachable rhodopsins retaining the full natural chromophore. J. Am. Chem. Soc. 1980, 102, 6370-6372. (19) Mao, B.; Tsuda, M.; Ebrey, T. G.; Akita, H.; Balogh-Nair, V.; Nakanishi, K. Flash photolysis and low temperature photochemistry of bovine rhodopsin with a fixed 11-ene. Biophys. J. 1981, 35, 543-546. (20) Tachibanaki, S.; Imai, H.; Mizukami, T.; Okada, T.; Imamoto, Y.; Matsuda, T.; Fukada, Y.; Terakita, A.; Shichida, Y. Presence of two rhodopsin intermediates responsible for transducin activation. Biochemistry 1997, 36, 14173-14180. (21) Yamashita, T.; Terakita, A.; Shichida, Y. Distinct roles of the second and third cytoplasmic loops of bovine rhodopsin in G protein activation. J. Biol. Chem. 2000, 275, 3427234279. (22) Rabiner, L. R. A Tutorial on Hidden Markov Models and Seleceted Applications in Speech Recognition. Proce. IEEE 1989, 77, 257-286. (23) Bronson, J. E.; Fei, J.; Hofman, J. M.; Gonzalez, R. L., Jr.; Wiggins, C. H. Learning rates and states from biophysical time series: a Bayesian approach to model selection and singlemolecule FRET data. Biophys. J. 2009, 97, 3196-3205. (24) Imamoto, Y.; Kataoka, M.; Tokunaga, F.; Palczewski, K. Light-induced conformational changes of rhodopsin probed by fluorescent Alexa594 immobilized on the cytoplasmic surface. Biochemistry 2000, 39, 15225-15233.


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(25) Kim, J. M.; Altenbach, C.; Kono, M.; Oprian, D. D.; Hubbell, W. L.; Khorana, H. G. Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 12508-12513. (26) Lamichhane, R.; Liu, J. J.; Pljevaljcic, G.; White, K. L.; van der Schans, E.; Katritch, V.; Stevens, R. C.; Wüthrich, K.; Millar, D. P. Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 14254-14259. (27) Vogel, R.; Siebert, F. Conformations of the active and inactive states of opsin. J. Biol. Chem. 2001, 276, 38487-38493. (28) Altenbach, C.; Kusnetzow, A. K.; Ernst, O. P.; Hofmann, K. P.; Hubbell, W. L. Highresolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7439-7444. (29) Farrens, D. L.; Altenbach, C.; Yang, K.; Hubbell, W. L.; Khorana, H. G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science 1996, 274, 768-770. (30) Vogel, R.; Sakmar, T. P.; Sheves, M.; Siebert, F. Coupling of protonation switches during rhodopsin activation. Photochem. Photobiol. 2007, 83, 286-292.


21


Relative Intensity

a

WT

C140S

1.4

C140S

M257Y

Meta-II +NH2OH (Meta-II → Opsin)

1.3 1.2

Meta-II

1.1

+NH2OH (Meta-II → Opsin)

1.0 0

50 100 150 200

0

Time (sec)

50 100 150 200 Time (sec)

b

10 µm

10 µm

c

0

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 27

0

0 0

5

10

15

20

Time (sec)

25

0

5

10

15

20

25

Time (sec)

Figure 1. Fluorescence measurements of Alexa594 immobilized at Cys316. (a) Bulk fluorescence measurements of WTC140S (left) and M257YC140S (right).

Photoactivation of

WTC140S and M257YC140S at t=0 generated Meta-II, which shows fluorescence increase (red). In the presence of 50 mM hydroxylamine, Meta-II decayed to opsin (blue). It should be noted that fluorescence intensity of WTC140S opsin was smaller than Meta-II, while that of M257YC140S opsin was comparable to Meta-II. (b) The TIRFM image of liposomes containing WTC140S (left) or M257YC140S (right) opsin (pH 6.0). (c) Typical time courses of fluorescence intensity of


22

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WTC140S (left) and M257YC140S opsin (right) at pH 6.0. Fluorescence change points were detected by using a laboratory-written analytical program based on a hidden Markov model (smooth orange/magenta/cyan lines). Intensity change at fluorescence change point (∆I, orange) and duration times of high fluorescence state (Fhigh, magenta) and low fluorescence state (Flow, cyan) were analyzed (Figures 2 and 3, respectively).


23


C140S

(a) WT

Frequency (count/s)

(b) M257Y

0.03 0.02 0.01 0.00 0.03 0.02 0.01 0.00 0.03 0.02 0.01 0.00 -0.8

-0.4

0.0

C140S

dark (n=1083)

dark (n=1161)

light (n=1221)

light (n=1385)

opsin (n=1016)

opsin (n=971)

0.4

0.8

Intensity Change (∆I)

-0.8

-0.4

0.0

0.4

0.8


(c) Denatured 0.03 0.02 0.01 0.00

(n=782)

-0.8

-0.4

0.0

0.4

0.8


(d)

Frequency (count/s)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 27

0.15 0.10 0.05 0.00 Dark Light Opsin Dark Light Opsin

WT

C140S

M257Y

C140S

Figure 2. The amplitude and frequency of fluorescence changes. (a-c) The histograms of difference in fluorescence intensity at the change points (∆I, orange lines in Figure 1c) in the dark state (dark), photoactivated state (light), and opsin (opsin) for WTC140S (a) and M257YC140S (b). The histogram for the denatured sample is also shown for comparison (c). Green and blue curves show signal components (SC) and noise components (NC), respectively, and black curves show the sum of SC and NC. (d) Frequency of the conformational change derived from the SC.


24

Page 25 of 27

0.3

C140S

(a) WT

C140S

Light

(c) M257Y

Opsin

(d) M257Y

Light

flow→high

0.2 0.1

fhigh→low

0.0 0.3

C140S

(b) WT

C140S

Opsin

0.2

Frequency (count/s)

0.1 0.0 0.3

0

(e) Boil

1

2

3

4

Time (sec) 0.2 0.1 0.0 0

1

2

3

4

Rate Constsnt (sec ) kS1 kS2

Time (sec) –1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(f)

active → inactive inactive → active

1.0

0.5

0.0

Light

Opsin WT

C140S

Light

Opsin C140S

M257Y

Figure 3. Histograms of duration times of Fhigh and Flow. (a-e) Histograms of duration time Fhigh (magenta in Figure 1c) and Flow (cyan in Figure 1c) were constructed for photoactivated state and opsin of WTC140S and M257YC140S (a-d) or boiled sample (e). (f) Rate constants for SC from Fhigh to Flow (kS1, red) and those from Flow to Fhigh (kS2, blue) for photoactivated state (light) and opsin were estimated by global fitting (Supporting Information). Error bar indicates the standard deviation of curve fitting.


25


Page 26 of 27

ligand free CAM

Opsin

Opsin* CAM ATR

ATR

11CR

Meta-II

Meta-II*

hν

Opsin*•Gt agonist bound

Meta-II*•Gt

inverse-agonist bound CAM

Rh

Rh*

Rh*•Gt

Figure 4. Population shift model for constitutive activity of rhodopsin. (Gray) In ligand free opsin, M257Y mutation shifts the conformational equilibrium towards the active form (Opsin*) by elevating the rate constant for formation of Opsin*. It has been reported that M257Y can pick up all-trans-retinal (ATR) as the exogenous ligand3, 8 to form Meta-II*. (Yellow) In agonist bound form (Meta-II), effect of CAM on the conformational equilibrium was not observed. (Red) In the dark state, infrequent formation of Rh* was observed in M257Y, while it was not sufficient to activate G-proteins. This mechanism would be applicable for general GPCRs which are activated by diffusible ligands.


26

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TOC Graphics


27

Shift in Conformational Equilibrium Induces Constitutive Activity of G

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